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. 2026 Jan 10;27:157. doi: 10.1186/s12864-025-12506-9

Genome-wide characterization of OFP family in soybean reveals the roles of GmOFP genes involved in plant height regulation and salinity tolerance

Xiao Wang 1,2, Wei Liu 1,2, Yubin Wang 1,2, Yanwei Zhang 1,2, Wei Li 1,2, Caijie Wang 1,2, Ran Xu 1,2, Xiaoyan Zhang 1,2, Juanjuan Zhang 3, Lifeng Zhang 1,2,✉
PMCID: PMC12879352  PMID: 41519697

Abstract

Background

Ovate Family Proteins (OFPs), a class of plant-specific transcription factors, play vital roles in development and abiotic stress tolerance. However, the OFP family in soybean (Glycine max) has not been extensively studied.

Results

In this study, a total number of GmOFP genes were identified in soybean genome (42 members, grouped into four clades). qRT-PCR analysis of GmOFPs under GA and salt treatments revealed that GmOFP1b, GmOFP2, and GmOFP7 may be involved in plant height development and salinity response, and were therefore chosen for further study. Overexpression of GmOFP1b in Arabidopsis reduced plant height by ~ 40% and shortened siliques. Furthermore, transgenic plants expressing GmOFP1b, GmOFP2, and GmOFP7 exhibited higher survival rates under salt stress at the seedling stage. Moreover, GmOFP1b interacts with GmKNOX4 proteins, and appears to play a dual role in salt tolerance in mature plants and negatively regulates the expression of GmGA20ox to control plant height.

Conclusions

Our findings offer a comprehensive genomic characterization of the OFP gene family in soybean, providing a molecular framework for elucidating the functions of these genes in plant height control and salt stress tolerance.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12864-025-12506-9.

Background

The Ovate Family Proteins (OFPs) are plant-specific proteins characterized by a conserved C-terminal OVATE domain of 70 amino acids [1]. The first OFP gene was identified in tomato, where a mutation leading to premature transcription termination resulted in pear-shaped fruit [2]. OFPs are widespread in the plant kingdom, with varying numbers across species: 19 in Arabidopsis thaliana, 31 in Oryza sativa, 31 in Solanum lycopersicum, 45 in Zea mays, 29 in Brassica rapa, 74 in Capsicum annuum, and 100 in Triticum aestivu [1, 3–7].

In Arabidopsis thaliana, AtOFP1 acts as a transcriptional repressor, directly suppressing AtGA20ox1, a key gene in gibberellin (GA) biosynthesis [8]. Overexpression of AtOFP1, AtOFP2, AtOFP4, AtOFP5, and AtOFP7 leads to similar phenotypes, including shortened aerial organs, ovate-shaped tissues, and reduced fertility [8–10]. Additionally, AtOFP5 negatively regulates the BLH1-KNAT3 complex, crucial for early embryo sac development [11]. AtOFP1 and AtOFP4 also participate in secondary cell wall formation by interacting with AtKNAT7 to enhance its transcriptional repression activity [10]. In rice, OsOFPs have been mainly reported in plant development regulation. For example, OsOFP1 influences plant architecture and seed morphology, while OsOFP1 and OsOFP8 modulate brassinosteroid (BR) responses [12–14]. OsOFP2 and OsOFP6 affect plant height, grain shape, and lateral root growth [13, 15]. Beyond Arabidopsis and rice, OFPs in radish (Raphanus sativus L.), potato (Solanum tuberosum L.), cucumber (Cucumis sativus L.) and melon (Cucumis melo L.) govern tuber and fruit morphology [16–18]. Recent studies also highlight their roles in stress responses, such as TaOPF29a-A in wheat (Triticum aestivum L.) drought tolerance and OFP1 in Prunus persica salt tolerance [7, 19].

Plant height, a critical agronomic trait, influences resource utilization, lodging resistance, and yield [20, 21]. The stem is consisted of nodes and internodes, and the plant height is controlled by stem elongation [22]. The development of dwarfing and semi-dwarfing cultivars improved the lodging resistance and crop yield in wheat, rice, maize and soybean in Green Revolution, and GA is known as “green revolution hormone” [23, 24]. GA stimulates stem elongation by enhancing cell division and expansion, playing crucial roles in plant growth and development [25, 26]. Among GA biosynthesis, the GA 20-oxidase (GA20ox) members catalyze the final conversion of GA precursors into bioactive GAs, and mutations in GA20ox often result in semi-dwarf phenotypes [27–29].

Soybean (Glycine max), a vital cash crop providing protein and oil, has long been a target for height reduction without compromising yield. GA plays a central role in regulating soybean plant height. For example, GmDW1 encodes an ent-kaurene synthase involved in height control, while GmLHY modulates GA biosynthesis and response genes [30, 31]. However, soybean production faces significant challenges from abiotic stresses, particularly high salinity, making it essential to uncover the molecular mechanisms of salt tolerance for breeding resilient cultivars [32, 33]. The functions of OFP genes in plants are well-documented [6, 34], however, this family is uncharacterized in soybean, and its relationship with GA-mediated plant height regulation has not been investigated. This study identifies the OFP gene family in soybean and characterizes the roles of selected members in regulating plant height and salt tolerance, establishing a foundation for future research into their functions in development and stress response.

Results

Identification and phylogenetic analysis of GmOFPs

We identified 42 soybean OFP genes in this study (Table 1; Table S1), which were named based on their evolutionary relationships in Fig. 1. The predicted proteins encoded by these GmOFP genes ranged from 152 to 414 amino acids in length, with molecular weights (MW) of 37.69–63.68 kDa and isoelectric points (pI) of 4.08–10.61. To investigate evolutionary relationships among GmOFP proteins and their homologs, we constructed a phylogenetic tree comprising 19 AtOFPs, 31 OsOFPs, and 42 GmOFPs (Fig. 1). Based on the phylogenetic analysis, all the 92 OFP proteins were classified into four clades: Group1 to Group4, which correspond to those in Arabidopsis and rice and are characterized by the fact that overexpression of the same subfamily frequently results in similar phenotypes [9, 35]. GmOFPs were more closely related to AtOFPs than to OsOFPs, reflecting phylogenetic divergence between species and dicot lineage conservation. Group1 was the largest subfamily, with 15 GmOFPs, followed by Group2 (11 members) and Group3 (12 members). Group4 was the smallest, with only four OFPs.

Table 1.

List of OFP family genes from soybean

Name Locus Isoelectric point Protein Length Molecular weight (kD)
GmOFP1a Glyma.05G162200 9.84 409 99.02
GmOFP1b Glyma.08G119800 9.09 413 99.40
GmOFP2 Glyma.01G200600 4.08 166 43.62
GmOFP3a Glyma.18G054200 9.66 385 96.29
GmOFP3b Glyma.11G159800 9.98 385 96.2
GmOFP3c Glyma.02G235600 8.73 383 95.49
GmOFP3d Glyma.14G203600 8.8 374 93.39
GmOFP3e Glyma.18G215300 10.37 377 93.79
GmOFP3f Glyma.09G276400 10.36 386 96.16
GmOFP3g Glyma.03G036600 10.61 387 97.08
GmOFP3h Glyma.01G131900 10.53 385 96.48
GmOFP4 Glyma.15G086301 10.21 369 88.73
GmOFP5 Glyma.13G225800 9.51 414 99.49
GmOFP6a Glyma.02G199300 9.52 169 42.23
GmOFP6b Glyma.10G077800 9.64 177 44.17
GmOFP6c Glyma.19G197800 9.48 204 51.86
GmOFP6d Glyma.03G200200 10.15 190 48.54
GmOFP7 Glyma.06G033000 10.18 252 61.16
GmOFP8a Glyma.05G230900 10.37 285 71.71
GmOFP8b Glyma.08G038400 10.5 293 73.85
GmOFP11 Glyma.20G147700 8.65 190 51.45
GmOFP11a Glyma.03G155600 6.23 250 64.46
GmOFP11b Glyma.19G157700 5.34 262 67.59
GmOFP11c Glyma.02G146400 6.24 204 52.39
GmOFP11d Glyma.10G027500 6.41 219 55.73
GmOFP13a Glyma.10G277300 5.03 251 63.09
GmOFP13b Glyma.20G112400 4.58 238 59.9
GmOFP13c Glyma.03G200100 4.82 283 74.23
GmOFP13d Glyma.19G197700 4.48 272 70.65
GmOFP14a Glyma.15G014500 7.32 249 63.43
GmOFP14b Glyma.07G006200 8.65 282 70.56
GmOFP15a Glyma.17G010000 7.41 152 37.69
GmOFP15b Glyma.07G263900 7.79 156 38.53
GmOFP15 Glyma.10G077700 4.31 267 68.14
GmOFP17 Glyma.14G203500 10.11 186 46.1
GmOFP18 Glyma.02G199500 4.28 276 70.11
GmOFP19a Glyma.11G180300 6.57 197 50.03
GmOFP19b Glyma.12G093200 6.95 195 49.3
GmOFP19d Glyma.13G314000 7.9 184 46.24
GmOFP19c Glyma.12G187500 8.13 186 46.77
GmOFP20 Glyma.02G235500 9.92 185 45.94
GmOFP26 Glyma.10G247600 8.65 180 48.36
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Fig. 1.

Fig. 1

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Phylogenetic tree construction and analysis of OFP transcription factors. An unrooted neighbor-joining tree was generated using MEGA7, based on OFP sequences from Arabidopsis, Oryza sativa, and Glycine max with 1000 bootstrap replicates

Gene structures and conserved motifs analysis

To further investigate the diversification of GmOFPs, we analyzed their gene structures by aligning coding sequences with corresponding genomic sequences. The exon/intron organization revealed that all GmOFPs contain one or two exons (Fig. 2a), indicating structural conservation within this gene family throughout evolution. Previous studies showed that OVATE protein contains an OVATE domain, which is also present in plants [34, 36]. Consistent with this, sequence alignment confirmed that the GmOFPs retain this conserved OVATE domain (Figure S1). To better understand the protein sequence characteristics of GmOFPs, we analyzed their conserved motifs using the online MEME tool. A total of 10 motifs (designated as motifs 1–10) were identified (Fig. 2b). While some motifs were shared among most members, others were unique to specific subclasses. For instance, motifs 1 and 2, located diffusely at the C-terminal, were common and showed similarity to the OVATE domain of OFP proteins. In contrast, motif 10 was exclusive to Group 2, whereas GmOFP17 and GmOFP20 (Group 4) contained only motif 1. Additionally, motifs 4, 5, 7, 8, and 9 were present in Group 1, while motif 6 was unique to Group 3. These findings implied conserved motifs within a subgroup indicate similar functions, while the divergent distribution of motifs across different groups reflects functional divergence among the GmOFPs.

Fig. 2.

Fig. 2

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The phylogenetic relationship, exon/intron structure, and motifs in GmOFP family. a Phylogenetic tree based on GmOFP protein sequences. b The gene structures of GmOFP genes. CDS, introns, and untranslated regions (UTRs) are indicated by yellow boxes, black lines, and green boxes, respectively. c Conserved motifs of GmOFPs. The motifs were indicated using the MEME online tool and represented by the boxes with different numbers and colors

Expression profile of GmOFP genes in different organs

Previous studies have shown that overexpression of AtOFP1, AtOFP2, AtOFP4, AtOFP5, and AtOFP7 induces similar phenotypic changes in plants. Additionally, when OsOFP2 was overexpressed, it led to reduced plant height, altered leaf morphology, modified seed shape, and changes in vascular bundle arrangement in the stems [1, 15]. Moreover, a study also showed that OFP1 increased salt tolerance in tomato [19]. To explore the functions of GmOFP1, 2, 4, 5, and 7 involved in plant height development and salt tolerance in soybean, we analyzed their expression profiles across six organs including leaf buds, flowers, pods, stems, leaves and roots by quantitative real-time PCR (qRT-PCR). The results revealed that GmOFP1a and GmOFP1b exhibited similar expression patterns, with notably high transcript levels in flowers and leaves (Fig. 3a, b), suggesting their specialized roles in the development of these tissues. Likewise, GmOFP4 and GmOFP5 were also predominantly expressed in flowers and leaves (Fig. 3d, e), further supporting their potential involvement in flower and leaf development. In contrast, GmOFP2 showed relatively low expression in pods and stems, while GmOFP7 had the lowest expression in stems and the highest expression in roots (Fig. 3c, f).

Fig. 3.

Fig. 3

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The expression profiles of the six GmOFP genes in different tissues examined by qRT-PCR. The expressions of GmOFP1a, GmOFP1b, GmOFP2, GmOFP4, GmOFP5 and GmOFP7 were shown in a-f, respectively. Bars represent means ± SD of three biological replicates

Expression profiles of GmOFPs in response to GA and salt stress

While roots direct growth by integrating environmental cues and coordinating stress responses, internal hormonal signals like GA are essential for regulating overarching developmental programs such as plant height [37, 38]. To investigate the response of the aforementioned genes to GA in soybean, we conducted qRT-PCR analysis on seedlings treated with GA3 at the root level. The results revealed that GmOFP1b expression was upregulated under GA3 treatment (Fig. 4b), whereas GmOFP7 was significantly downregulated at 1 h. In contrast, GmOFP4 and GmOFP5 showed decreased expression only after 6 h (Fig. 4d-f). Meanwhile, GmOFP1a and GmOFP2 showed no response to GA (Fig. 4a, c). These results indicated that GmOFP1b and GmOFP7 were induced by GA in roots and may be more promising candidates for further studies on plant height regulation. To test the response of these GmOFPs to salt in soybean, qRT-PCR analyses were performed on seedling treated with NaCl at 4 time points. These results displayed that the expressions of 6 GmOFP genes were continuously induced by salt (Fig. 4g-l), and the expression of GmOFP1b, GmOFP2 and GmOFP7 were represented the most significant change under NaCl (Fig. 4h, I, l), being the best candidate for further study.

Fig. 4.

Fig. 4

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The expression of the GmOFP genes under GA3 and NaCl treatment. a-f The expression of the six GmOFP genes under GA3 treatments. g-l The expression of the six GmOFP genes under NaCl treatments. Bars represent means ± SD of three biological replicates. Student’s T-test was used to examine the statistical significance (*** p < 0.001, ** p < 0.01, *p < 0.05)

Ectopic expression of GmOFP1b in Arabidopsis reduced the plant height and silique length

To investigate the role of GmOFP1b, GmOFP2 and GmOFP7 in plant development or salt tolerance, these three genes were overexpressed in Arabidopsis, respectively. The transgenic lines were generated, and qRT-PCR analyses showed that their transcriptional levels were significantly increased in transgenic plants, and those with the highest expression levels compared to the wild type were selected for phenotypic analysis and NaCl treatment assays (Figure S2). Phenotypic observations revealed that 30-day-old 35 S: GmOFP1b-3 plants exhibited reduced plant height compared to wild type, whereas 35 S: GmOFP2-2 and 35 S: GmOFP7-4 plants showed no significant difference in height (Fig. 5a, b). The other two 35 S: GmOFP1b transgenic lines also exhibited a dwarf phenotype, whereas the 35 S: GmOFP2 and 35 S: GmOFP7 lines were similar to the wild type (Figure S3 a, b). Subsequently, shortened siliques were observed only in 35 S: GmOFP1b-3 plants, and measurement results revealed that the silique length of the 35 S: GmOFP1b-3 overexpression lines was significantly shorter than that of the wild type (WT) (Fig. 5c, d). These results suggested that GmOFP1b overexpression may slow plant growth, while GmOFP2 and GmOFP7 do not appear to affect this trait under normal conditions.

Fig. 5.

Fig. 5

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Phenotypic analysis of GmOFP1b/2/7-overexpressing transgenic plants and the wild type (WT). a An overview of 30-day-old seedlings of the 35 S: GmOFP1b/2/7 transgenic lines and WT plants. b Plant height of transgenic lines and WT plants. Values with error bars represent mean ± SD (n = 9). c Siliques of the transgenic lines and WT plants. d Silique length of transgenic lines and WT plants. Values with error bars represent mean ± SD (n = 9). Student’s T-test was used to examine the statistical significance (*** p < 0.001). Bars: 1 cm (a), 5 mm (c)

GmOFP1b negatively regulates GmGA20ox in plant height regulation

Previous studies have shown that AtOFP1 suppresses cell elongation by transcriptionally repressing AtGA20ox1, while OsOFP2 alters seed shape and leaf morphology by downregulating OsGA20ox7 expression [8, 15]. To investigate the regulatory relationship between GmOFP1b and GmGA20ox, we first check the expression levels of AtGA20ox genes in wild type and 35 S: GmOFP1b-1, −2, −3 lines. Result showed that all the AtGA20ox genes were significantly decreased in all GmOFP1b overexpressing lines except AtGA20ox1 (Fig. 6a). This result indicated that GmOFP1b negatively regulates GA20ox genes expression in plant height control. To further examine the regulation relationship, we conducted dual-luciferase reporter (DLR) assays (Fig. 6b-i). Phylogenetic analysis identified nine GmGA20ox genes in soybean (Figure S4), six of which were selected for DLR assays. The luciferase reporter construct driven by the GmGA20ox promoter was co-transformed with GmOFP1b effector proteins into tobacco leaves. The results revealed that decreased luciferase luminescence for 35 S: GmOFP1b-GFP and pGmGA20ox1a/1b/2a: LUC co-transformants compared to control, in contrast, no significant change in luminescence was observed in the 35 S: GmOFP1b-GFP and pGmGA20ox2b/3/4:LUC co-transformants, indicating GmOFP1b specifically suppresses the promoters of GmGA20ox1a, GmGA20ox1b and GmGA20ox2a in vivo (Fig. 6b-i).

Fig. 6.

Fig. 6

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GmOFP1b negatively regulates GmGA20ox to control plant height. a The expression levels of AtGA20ox family in WT and 35 S: GmOFP1b transgenic lines determined by qRT-PCR. b Schematic representation of reporter and effector constructs used in transient expression assay. c-i Representative bioluminescence images of the DLR assay. The pGmGA20ox: LUC reporter constructs were transiently infiltrated in N. benthamiana leaves together with the 35 S: GmOFP1b-GFP effector. The pseudocolor bar shows luminescence intensity. GFP effector was used as negative control. Bars represent means ± SD of three biological replicates, and statistical significance was determined using Student’s T-tests: ***, P < 0.001

Ectopic expression of GmOFPs in Arabidopsis improved the plant salt tolerance

To investigate the role of GmOFPs in salt stress, 35 S: GmOFP1b-3, 35 S: GmOFP2-2 and 35 S: GmOFP7-4 plants were used to perform NaCl treatment assays. Under normal conditions, the leaves of 35 S: GmOFP1b-3 were smaller, and the general growth status of transgenic plants were comparable to that of wild type plants (Fig. 7a). However, following a 20-day treatment with 250 mM NaCl, independent 35 S: GmOFP1b transgenic lines exhibited stronger growth and greener leaves than the wild type and 35 S: GmOFP2 and 35 S: GmOFP7 transgenic lines (Fig. 7b-d, Figure S3 c, d). Quantification of chlorophyll content and survival rate revealed that the 35 S: GmOFP1b-3 line was the most robust, displaying significantly higher values than both the wild type and other transgenic lines (Fig. 7e, f), suggesting a particularly strong role for GmOFP1b in salt tolerance at the mature plant stage. This enhanced tolerance was also observed at the seedling stage. When plants grown on MS agar, the roots of 35 S: GmOFP1b-3 were relatively shorter (Fig. 7g), when the MS agar supplemented with 150 mM NaCl, all three transgenic seedlings were greener and had higher survival rates than the wild type (Fig. 7h). These results collectively indicate that GmOFPs, especially GmOFP1b, positively regulate salt stress tolerance across multiple developmental stages.

Fig. 7.

Fig. 7

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The phenotype of the 35 S:GmOFP transgenic Arabidopsis plants under NaCl treatment. a-d The phenotype of WT and 35 S: GmOFP transgenic Arabidopsis grown in soil under normal conditions or under 250 mM NaCl treatment. e and f The chlorophyll content and survival rate of the transgenic and WT plants grown under normal conditions or under 250 mM NaCl treatment. g and h An overview (g) and the survival rate (h) of the WT seedlings and the transgenic Arabidopsis seedlings grown in 1/2 MS with or without 150 mM NaCl. Bars represent means ± SD of three biological replicates, and statistical significance was determined using Student’s T-tests: ***, P < 0.001. Bars: 5 cm (a- d), 1 cm (g)

Interaction analysis of GmOFPs with GmKNOX4 proteins

Previous studies have demonstrated that OFP proteins interact with KNAT7 homologs to form protein complexes involved in regulating various biological processes [10, 15, 39]. To investigate whether the three GmOFP proteins interact with KNAT7 homologs in soybean, we identified four candidate proteins named GmKNOX4a, GmKNOX4b, GmKNOX4c, and GmKNOX4d through phylogenetic analysis (Figure S5). Using Yeast Two-Hybrid (Y2H) assays, we examined the interactions between GmOFPs and GmKNOX4s. The results revealed strong interactions between GmOFP1b and all four GmKNOX4 proteins (GmKNOX4a-d), while weaker interactions were detected between GmKNOX4b and GmOFP2/GmOFP7 (Fig. 8). These data indicated the existence of interaction specificity between OFP and KNAT7/KNOX4 proteins.

Fig. 8.

Fig. 8

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Identification of the interactions between GmOFP1b/2/7 and GmKNOX4 proteins by yeast two-hybrid assay (Y2H). All transformants were able to grow on double dropout (DDO) medium (SD/-Leu/-Trp). Protein-protein interactions were confirmed by yeast colonies that grew on triple dropout (TDO) medium (SD/-His/-Leu/-Trp) containing X-α-Gal and exhibited blue coloration

Discussion

OFP genes, which encode a class of plant-specific transcriptional repressor, have been identified in numerous plant species [3, 34, 40–42]. However, limited information is available on OFP genes in soybean. In this study, we identified 42 GmOFP genes, classified into four groups based on phylogenetic analysis with Arabidopsis and rice. The number of GmOFP genes in soybean exceeds that in Arabidopsis, rice, Populus trichocarpa, Betula luminifera, and Solanum lycopersicum [5, 36, 39]. The differences in OFP gene number and density between species might due to gene duplication events or the genome size [43, 44]. Gene structure serves as a key indicator of gene function and classification, with intron gain or loss reflecting evolutionary selection pressures [45]. Notably, only four GmOFP genes contain a single intron, while the rest are intron-free, consistent with findings in other species where OFP genes typically possess no more than two introns. This further supports their close evolutionary relationship. Motifs and domains are key structural and functional elements of proteins or protein families, playing critical roles in various regulatory processes such as protein-protein interactions, transcriptional activity, and DNA binding [46, 47]. In our study, we identified 10 conserved motifs. While individual GmOFP may exhibit variations in motif composition, members within the same subgroup generally share similar motif patterns. These findings demonstrate a strong correlation between motif composition and phylogenetic grouping. Additionally, an OVATE domain was detected at the C-terminus of all GmOFPs, consistent with previous reports [3, 9, 34, 48].

OFP genes play diverse roles in various stages of plant growth and development [34, 49], including the critical function of modulating plant height. For example, overexpression of the OsOFP2 gene and OsOFP6 led to reduced plant height [13, 15], likewise, overexpression of BlOFP3 and BlOFP5 genes showed shorter plants than wild plants [39]. These findings suggest that OFP genes, including GmOFP1b, are key regulators of plant architecture. Consistent with this, our study demonstrated that GmOFP1b overexpression in Arabidopsis inhibits silique elongation, a phenotype shared with its homologs AtOFP1, RsOFP2.3, CmOFP13 (Cucumis melo) and BhiOFP1 (Benincasa hispida) [18, 41, 50]. This further supports the conserved role of OFP genes in controlling organ elongation across species. Notably, among the tested GmOFPs, only GmOFP1b overexpression led to dwarfism and shortened siliques, whereas GmOFP2 and GmOFP7 did not. This difference likely stems from their classification into distinct OFP subfamilies, as members of the same subfamily often induce similar phenotypes when overexpressed. For example, Class I OFP genes overexpression result in kidney-shaped cotyledons and curled leaves, Class II leads to thick, flat, and cyan leaves, and Class III causes blunt-ended siliques [1].

Studies across various plant species have established that OFP genes are key regulators of growth and development, integrating phytohormone signaling and environmental stress responses. AtOFP1 functions in GA signaling by repressing the expression of GA20ox1, a key gene in GA biosynthesis in Arabidopsis [8]. In contrast, most reported OsOFPs in rice are associated with the hormone BR and show normal responses to GA treatment [42, 51, 52]. Similar to AtOFP1, GmOFP1b is induced by GA and represses the GmGA20ox expression. This highlights a conserved function for OFPs in the GA signaling pathway in Arabidopsis and soybean, alongside functional diversity across species. Furthermore, OFP genes enhance drought tolerance in wheat and Populus trichocarpa [7, 36], and salt tolerance in tomato [19]. We extend this conserved function by demonstrating that GmOFP1b is a positive regulator of salt tolerance in soybean (Fig. 9). GmOFP1b, along with GmOFP2 and GmOFP7, responds to salt stress. Additionally, GmOFP1b and GmOFP7 are also responsive to GA. These indicate that GmOFP gene is likely regulated by both environmental signals and phytohormones.

Fig. 9.

Fig. 9

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Model for the proposed roles of GmOFP1b in Glycine max. GmOFP1b is likely to repress GmGA20ox expression to control plant height, potentially via interaction with GmKNOX4. Moreover, GmOFP1b functions as a positive regulator of salt tolerance

It has been previously demonstrated that a subset of OFP proteins engages in physical interactions with specific members of the KNOX and BLH families of TALE homeodomain proteins [53]. OFP proteins do not contain DNA-binding domains, but OFP1 functions as a transcriptional repressor, a model was proposed that OFPs interact with DNA-binding partners like KNOX or BLH proteins to form a functional repression complex that controls development [8, 11]. For example, secondary cell wall formation is regulated by a functional complex of the KNOX protein KNAT7 with OFP1 or OFP4 [10, 54], AtOFP5 interacts with KNAT3 and BLH1 to suppress the activity of BELL-KNOX complexes, which is essential for the development of the Arabidopsis embryo sac [11]. Likewise, OsOFP19 forms a complex with Oryza sativa homeobox1 (OSH1) that enhances their transcriptional activity [51]. In addition, OsOFP9 interacts with GS9 and Dwarf and low-tillering (DLT), interactions which also influence protein activity [52]. Accordingly, although further experiments are needed for confirmation, we have demonstrated that GmOFPs interact with the GmKNOX4/KNAT7 family. This interaction is supported by evidence that OsKNAT7 overexpression also reduces plant heigh [55]. Based on this, we propose a model that the GmOFP-GmKNOX4 complex may affect their activity. Alternatively, GmOFP1b is likely to repress GmGA20ox expression, potentially via interaction with GmKNOX4 (Fig. 9). This is consistent with the fact that GmOFP1b, which lacks a DNA-binding domain, likely regulates GmGA20ox indirectly through this protein-protein interaction. Moreover, this module is further substantiated by the demonstrated ability of KNOX proteins to bind the GA20ox promoter [56, 57].

The dramatic yield increases of the Green Revolution were directly attributable to the adoption of semi-dwarf varieties, which offered superior lodging resistance and permitted higher planting densities. Today, advancing abiotic stress tolerance through crop breeding represents an equally critical strategy for further improving crop productivity. Given the dual function of GmOFP genes in modulating plant architecture and salt stress response, they represent valuable genetic targets for engineering soybean varieties capable of high-density cultivation in saline conditions.

Conclusions

We provide a comprehensive analysis of the OFP gene family in soybean, elucidating gene structures, expression patterns, and functional implications for salinity tolerance and plant architecture. The results indicated that GmOFP1b represses GmGA20ox expression, potentially via interaction with GmKNOX4. GmOFP1b, GmOFP2, and GmOFP7 all conferred salinity tolerance to seedlings. Notably, GmOFP1b also enhanced tolerance in mature plants. This research paves the way for breeding soybean varieties that are more tolerant to salt and higher yielding at greater planting densities, thereby supporting global food security and sustainable farming practices.

Materials and methods

Plant material, growth conditions and soybean treatments

The cultivar Williams 82 was used in this study. All the plants were cultivated in a growth chamber at 22 °C ± 2 °C under 16-h light and 8-h dark, with a relative humidity of 50%–60%. In the hydroponics experiment, soybean seeds were initially germinated in sand for 3–4 days, uniform seedlings were then transferred to a container filled with half-strength Hoagland’s solution, which was replaced every three days until the emergence of the first trifoliate leaf. Later, the soybean seedlings were treated with 150 mM NaCl or 10 µM GA₃ at specified time points. Roots from the treated seedlings were collected at 0, 1, 3, and 6 h, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent RNA extraction and qRT-PCR analysis. Three biological replicates were conducted, and a T-test was used to evaluate the statistical significance of observed differences.

Identification of OFPs and phylogenetic analysis

To identify OFP Proteins in soybean, we performed a BLASTP search against the Glycine max Wm82.a4.v1 genome assembly version in Phytozome (https://phytozome-next.jgi.doe.gov/), using 19 known Arabidopsis thaliana OFP proteins as queries. Candidate sequences with an e-value below 1e-10 were retained and validated using the SMART online database (https://smart.embl.de/). Only the most complete transcripts were selected for further analysis, while splice variants were excluded. To examine the phylogenetic relationships among OFP proteins, we constructed a phylogenetic tree using 19 Arabidopsis thaliana OFP proteins, 31 Oryza sativa OFP proteins, and the 42 identified soybean OFP proteins. The tree was generated in MEGA7 using the Neighbor-Joining method with 1000 bootstrap replications for reliability assessment. The Arabidopsis thaliana OFP (AtOFP) amino acid sequences were retrieved from The Arabidopsis Information Resource (TAIR) database (http://www.arabidopsis.org/), while the Oryza sativa OFP (OsOFP) sequences were obtained from the Rice Genome Annotation Project (http://rice.plantbiology.msu.edu/) and the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/). Similarity, the amino acid sequences of the GA20ox and KNAT7/KNOX4 homologs were obtained through BLAST searches in the Phytozome database, and phylogenetic trees were generated using the neighbor-joining method in MEGA 7.0 with 1000 bootstrap replicates.

Gene structure and conserved motifs analysis

The exon-intron structures of GmOFP genes were analyzed by aligning their coding sequences (CDS) with corresponding genomic DNA sequences using the Gene Structure Display Server (GSDS 2.0, http://gsds.gao-lab.org/). To identify conserved motifs in GmOFP proteins, we utilized the online MEME suite (http://meme-suite.org/tools/meme). Protein sequences were aligned using Clustal X2, and homology shading was performed with GeneDoc software.

Overexpression of GmOFPs in Arabidopsis

The full-length coding sequences (CDS) of GmOFP2 and GmOFP7 were amplified and cloned into the pENTR/D-TOPO vector (Invitrogen). These constructs were then recombined into the destination vector pEarleyGate 103 using Gateway LR Clonase (Invitrogen). Meanwhile, the GmOFP1b CDS was amplified and inserted into the pCAMBIA1300 vector to generate 35 S: GmOFP1b-GFP plasmid. All constructs were transformed into Arabidopsis (Col-0) via Agrobacterium tumefaciens strain GV3101. The transgenic seedlings selected on antibiotic-containing MS plates were confirmed by qRT-PCR using leaves, and homozygous T3 or T4 plants from independent lines were used for all phenotypic analyses and subsequent experiments. The primers used for PCR and qRT-PCR are listed in Table S2.

RNA extraction and gene expression analysis

To examine the expression levels of GmOFP genes across various tissues, samples of leaf buds, leaves, flowers, stems, pods and roots were collected from Williams 82. Total RNA was extracted from these tissues using Trizol-RT Reagent (Molecular Research Center, Inc.) according to the manufacturer’s protocol. RNA extraction, cDNA synthesis, and qRT-PCR analyses were conducted as previously described [44]. Briefly, qRT-PCR was performed using a Roche 480 Light Cycler with Takara SYBR Premix Ex Taq. Gene expression was calculated using the 2^−ΔΔCT method [58]. For profiling GmOFP genes in soybean tissues and under treatments, GmActin (Glyma.18G290800) served as the reference gene. In contrast, AtActin2 (AT3G18780) was used as the reference for analyzing GmOFP expression in transgenic Arabidopsis [44]. Each sample had three biological replicates and the primers used are provided in Table S2. Statistical comparisons between different sample groups were performed using the T-test.

DLR assay

The 35 S: GmOFP1b-GFP vector was used as the effector. The 1.9∼2.2-kb promoter fragments upstream of GmGA20ox were cloned into the pGreenII-0800-Luc vector to generate the reporters. The effector and reporter constructs were co-introduced into Agrobacterium tumefaciens GV3101 with the helper plasmid pSoup19. Equal volumes of the bacterial suspensions containing the effector or reporter were co-infiltrated into tobacco leaves. After 48 h of incubation under light, luciferase activity was detected by spraying the leaves with a 1 mM d-luciferin solution (GoldBio), incubating them in darkness for 5 min, and capturing the luminescence with a Tanon 5200 plant live imaging system. The primers used in this study are provided in Table S2.

Salt stress treatment of the transgenic Arabidopsis

For the NaCl tolerance assay, T3 transgenic Arabidopsis plants were germinated on half-strength MS medium. Three-day-old seedlings were transferred to fresh medium containing either 150 mM NaCl or no NaCl (control). After five days, the survival rate was calculated from at least 16 seedlings. In a parallel soil-based assay, three-day-old seedlings were transplanted into soil and grown for two weeks under long-day conditions before being treated with 250 mM NaCl for 20 days, and survival was assessed using at least 12 plants. Total chlorophyll from leaves of wild type and transgenic plants were isolated in 90% acetone (v/v). The chlorophyll content was calculated spectrophotometrically as described by previous paper [59]. All experiments included three biological replicates, and statistical significance was evaluated using a T-test.

Yeast two-hybrid assay

The yeast two-hybrid (Y2H) assay was conducted using the Matchmaker Gold System (Clontech) following previously described methods [60]. The full-length CDS of GmKNOX4 were PCR-amplified and individually cloned into the pGBKT7 vector to generate the bait plasmids (GmKNOX4a-BD, GmKNOX4b-BD, GmKNOX4c-BD, and GmKNOX4d-BD). Moreover, the full-length CDS of GmOFP1b was PCR-amplified and cloned into the pGADT7 vector to construct the prey plasmid (GmOFP1b-AD). The bait and prey constructs were co-transformed into the yeast strain AH109 and plated on SD/-Trp/-Leu medium, followed by incubation at 30 °C for 3–5 days. Putative protein-protein interactions were assessed by growing the transformants on selective SD/-Leu/-Trp/-His/X-α-gal medium.

Supplementary Information

12864_2025_12506_MOESM1_ESM.tif (4.6MB, tif)

Supplementary Material 1: Figure S1. OVATE domain alignment of GmOFP proteins. The location of the OVATE domain (underlined) was indicated. 

12864_2025_12506_MOESM2_ESM.tif (3.5MB, tif)

Supplementary Material 2: Figure S2. Transgenic Arabidopsis plants confirmation with qRT-PCR. Expression analysis of GmOFP1b, GmOFP2 and GmOFP7 in 35 S: GmOFP1b/2/7 transgenic lines by qRT-PCR, respectively.

12864_2025_12506_MOESM3_ESM.tif (13.3MB, tif)

Supplementary Material 3: Figure S3. Phenotypic characterization of GmOFP1b, GmOFP2, and GmOFP7 overexpressing transgenic lines.

12864_2025_12506_MOESM4_ESM.tif (505.8KB, tif)

Supplementary Material 4: Figure S4. Phylogenetic tree analysis of GA20ox putative orthologs in Arabidopsis and Glycine max. A neighbor-joining phylogenetic tree was constructed and the bootstrap test was set to 1,000 replicates.

12864_2025_12506_MOESM5_ESM.tif (222.4KB, tif)

Supplementary Material 5: Figure S5. Phylogenetic tree analysis of KNOX4/KNAT7 putative orthologs in Arabidopsis, Medicago truncatula, Glycine max, Solanum lycopersicum, and Oryza sativa. The neighbor-joining phylogenetic tree was constructed and the bootstrap test was set to 1,000 replicates.

12864_2025_12506_MOESM6_ESM.xlsx (15KB, xlsx)

Supplementary Material 6: Table S1. Sequences of OFP proteins in soybean.

12864_2025_12506_MOESM7_ESM.xlsx (12.9KB, xlsx)

Supplementary Material 7: Table S2. Sequences of primers used in this study.

12864_2025_12506_MOESM8_ESM.xlsx (22.8KB, xlsx)

Supplementary Material 8: Table S3. Table S3 Raw data for this paper. The raw data including the CT values, plant height and silique length, chlorophyll content and survival rate.

Acknowledgements

We would like to express our sincere gratitude to all our colleagues in the paper.

Authors’ contributions

XW conceived the project; XW and LZ designed the experiments; XW, WL, YW, YZ, WL, CW performed the experiments; RX, XZ analyzed data; XW, JZ and LZ wrote the manuscript. All authors reviewed the manuscript.

Funding

This work was supported by Agricultural Variety Improvement Project of Shandong Province (2024LZGC030), Taishan Scholars Program (tsqnz20240845), National Natural Science Foundation of China (32300691, 32400683) and of Shandong Province (ZR2023QC178, ZR2024QC201), Agricultural Scientific and Technological Innovation Project, Shandong Academy of Agricultural Sciences.

Data availability

The protein sequences of GmOFPs were obtained from the database Phytozome ([https://phytozome-next.jgi.doe.gov/](https://phytozome-next.jgi.doe.gov)) and were shown in Table S1.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Change history

2/20/2026

The original publication was amended to correct formatting errors in the references.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

12864_2025_12506_MOESM1_ESM.tif (4.6MB, tif)

Supplementary Material 1: Figure S1. OVATE domain alignment of GmOFP proteins. The location of the OVATE domain (underlined) was indicated. 

12864_2025_12506_MOESM2_ESM.tif (3.5MB, tif)

Supplementary Material 2: Figure S2. Transgenic Arabidopsis plants confirmation with qRT-PCR. Expression analysis of GmOFP1b, GmOFP2 and GmOFP7 in 35 S: GmOFP1b/2/7 transgenic lines by qRT-PCR, respectively.

12864_2025_12506_MOESM3_ESM.tif (13.3MB, tif)

Supplementary Material 3: Figure S3. Phenotypic characterization of GmOFP1b, GmOFP2, and GmOFP7 overexpressing transgenic lines.

12864_2025_12506_MOESM4_ESM.tif (505.8KB, tif)

Supplementary Material 4: Figure S4. Phylogenetic tree analysis of GA20ox putative orthologs in Arabidopsis and Glycine max. A neighbor-joining phylogenetic tree was constructed and the bootstrap test was set to 1,000 replicates.

12864_2025_12506_MOESM5_ESM.tif (222.4KB, tif)

Supplementary Material 5: Figure S5. Phylogenetic tree analysis of KNOX4/KNAT7 putative orthologs in Arabidopsis, Medicago truncatula, Glycine max, Solanum lycopersicum, and Oryza sativa. The neighbor-joining phylogenetic tree was constructed and the bootstrap test was set to 1,000 replicates.

12864_2025_12506_MOESM6_ESM.xlsx (15KB, xlsx)

Supplementary Material 6: Table S1. Sequences of OFP proteins in soybean.

12864_2025_12506_MOESM7_ESM.xlsx (12.9KB, xlsx)

Supplementary Material 7: Table S2. Sequences of primers used in this study.

12864_2025_12506_MOESM8_ESM.xlsx (22.8KB, xlsx)

Supplementary Material 8: Table S3. Table S3 Raw data for this paper. The raw data including the CT values, plant height and silique length, chlorophyll content and survival rate.

Data Availability Statement

The protein sequences of GmOFPs were obtained from the database Phytozome ([https://phytozome-next.jgi.doe.gov/](https://phytozome-next.jgi.doe.gov)) and were shown in Table S1.

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